Free thyroxine is the generally accepted marker for the initial assessment of possible thyroid dysfunction: J. Clin. Invest., 45:133, 1966. Since approximately 0.05% of the total circulating thyroxine is physiologically active (i.e., free thyroxine), it is apparent that measurement of total thyroxine concentration only is not a completely satisfactory marker. Free thyroxine is however difficult to measure directly: Berger, S., and Quinn, J. L., "Fundamentals of Clinical Chemistry", Tietz, N. W., Ed; W. B. Saunders, Co.: Philadelphia, 1976, Chapter 14 and Robbins, J., "Thyroid Hormone Metabolism", Proceedings of an International Symposium at Glasgow, Harlem and Orr, Ed., 1975, Chapter 1. The remaining circulating thyroxine is bound to protein, primarily thyroxine binding globulin (TBG): Robbins, J., and Rall, J. G., Proteins Associated with Thyroid Hormones, Physiol. Rev., 40, 415, 1960. Thus, the measurement of total circulating thyroxine concentration and of thyroxine binding globulin concentration provides a good assessment of the relative concentration of free thyroxine.
Three different methods have been used to assess the level of thyroxine binding globulin concentrations. The oldest method is the T-3 uptake study which measures unsaturated thyroxine binding globulin capacity: J. Clin. Endocrinol., 17:33, January, 1957 and J. Clin. Endocrin., 25:39-54, 1965. T-3 uptake assays use .sup.125 I labeled liothyronine (T-3) to competitively bind to the free TBG sites and a secondary solid support (viz: resin, charcoal, etc.). After an incubation period and suitable separation technique (Viz: washing, centrifugation, etc.) the remaining radio-activity on the solid support is counted. This radioactivity is inversely proportional to the unsaturated thyroxine binding globulin concentration.
Another methodology is a radioimmunoassay which utilizes antibody specific for thyroxine binding globulin. This methodology does not measure the binding ability of the thyroxine binding globulins: Clin. Chem. Acta., 87, (1978), 373-381.
Another methodology assesses the total thyroxine binding globulin capacity by equilibrating with a large excess of thyroxine to displace the endogenous thyroxine in the sample: U.S. Pat. No. 3,960,492, (1976).
The present invention is a method for determining unsaturated thyroxine binding globulin in serum which has the particular advantage that the determination of unbound tracer, i.e., the thyroxine irreversible enzyme inhibitor conjugate, is sufficiently fast and irreversible so that thyroxine and TBG equilibrium is not disturbed.